Peptide-presenting surfaces for long-term culture of pluripotent cells

ABSTRACT

The present invention relates to methods of growing and maintaining pluripotent cells on an insoluble substrate that presents a peptide that binds to glycosaminoglycans, such as heparin. Specifically, methods of growing and maintaining pluripotent cells on substrates having a chemically defined surface presenting at least one peptide having basic amino acid residues separated by one or two hydrophobic amino acid residues.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/098,703 filed Sep. 19, 2008, incorporated herein byreference as if set forth in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States government support awarded bythe following agencies:

NIH A1055258. The United States government has certain rights in thisinvention.

BACKGROUND

The invention relates generally to culturing pluripotent cells, and moreparticularly to chemically defined (i.e., synthetic) surfaces forlong-term growth and maintenance (i.e., self-renewal) of pluripotentcells.

Pluripotent cells, such as embryonic stem cells (ESCs) and inducedpluripotent stem cells (iPS cells), have at least two characteristicsthat distinguish them from other types of cells. The firstcharacteristic is that they are self-renewing, and thus are capable ofgrowing indefinitely without differentiating. The second characteristicis that they can differentiate into cells of all three germ layers(i.e., endoderm, mesoderm, and ectoderm). See, e.g., Evans M & KaufmanM, “Establishment in culture of pluripotential cells from mouseembryos,” Nature 292:154-156 (1981), incorporated herein by reference asif set forth in its entirety.

One difficulty in working with pluripotent cells is developingstandardized culture conditions for these cells without requiring theuse of animal products or products such as serum, which tend to varyfrom batch to batch, to maintain the characteristics noted above.Important aspects of culturing these cells, therefore, are not only themedium in which they are grown, but also the surface upon which they arecultured.

Of particular interest herein are surfaces for culturing pluripotentcells, as these cells require adhesion/attachment to a surface tomaintain the characteristics noted above. Although much information isavailable on chemically defining the constituents for culture medium forthese cells, considerably less information is available on chemicallydefining the constituents of the surfaces and cell-substrate attachmentfor their survival and growth.

Initially, pluripotent cells were cultured on gelatin-coated surfacescontaining mouse embryonic fibroblasts (MEFs) or other feeder cells.See, e.g., Amit M, et al., “Human feeder layers for human embryonic stemcells,” Biol. Reprod. 68:2150-2156 (2003); Lee J, et al., “Establishmentand maintenance of human embryonic stem cell lines on human feeder cellsderived from uterine endometrium under serum-free condition,” Biol.Reprod. 72:42-49 (2005); and Thomson J, et al., “Embryonic stem celllines derived from human blastocysts,” Science 282:1145-1147 (1998).Pluripotent cells, however, do not grow on top of feeder cells, butinstead tend to occupy the exposed gelatin-coated surface. As the cellsproliferate, the growing colony pushes the MEFs away. See, e.g., ImrehM, et al., “Culture and expansion of the human embryonic stem cell lineHS181, evaluated in a double-color system,” Stem Cells Dev. 13:337-343(2004).

The art recognized that pluripotent cells can be cultured on agelatin-coated surface in the presence of secreted factors from feedercells, allowing the cells to be cultured in the absence of feeder celllayers (i.e., feeder-free). For example, feeder cell layers can beavoided through the use of “conditioned medium” (CM), which is medium inwhich feeder cells were cultured. However, culture of pluripotent cellson gelatin-coated surfaces in CM can lead to rapid differentiation ofthe cells. See, e.g., Xu C, et al., “Feeder-free growth ofundifferentiated human embryonic stem cells,” Nat. Biotechnol.19:971-974 (2001).

More recently, the art recognized that feeder cell layers also can beavoided by using a chemically defined culture medium (i.e., a completemedium), in which each constituent of the medium is fully disclosed andcharacterized. See, e.g., Ludwig T, et al., “Feeder-independent cultureof human embryonic stem cells,” Nat. Methods 3:637-646 (2006); andLudwig T, et al., “Derivation of human embryonic stem cells in definedconditions,” Nat. Biotechnol. 24:185-187 (2006), each of which isincorporated herein by reference as if set forth in its entirety.

One should not, however, overlook the role of surface attachment forsuccessful pluripotent cell maintenance and growth. In this regard,feeder cell layers can be avoided through the use of a commerciallyproduced extracellular matrix (ECM) material, such as Matrigel®.Matrigel®, however, contains indeterminate (i.e., undefined) quantitiesof murine extracellular matrix proteins, such as laminin, collagen andentactin. Additionally, there is batch to batch variation withinMatrigel® and other unknown components such as, growth factors. OtherECM materials that can be used for pluripotent cell culture includevitronectin, fibronectin and laminin.

Chemically defined surfaces for pluripotent cells have been described(see, e.g., Derda R, et al., “Defined substrates for human embryonicstem cell growth identified from surface arrays,” ACS Chem. Biol.2:347-355 (2007); and Gerecht S, et al., “Hyaluronic acid hydrogel forcontrolled self-renewal and differentiation of human embryonic stemcells,” Proc. Natl. Acad. Sci. USA 104:11298-11303 (2007), but thesesurfaces have not yet proven effective for long-term growth andmaintenance of pluripotent cells. Specifically, cells grown on thesesurfaces for several weeks form heterogeneous cell populations ofundifferentiated and differentiated cells, which can be challenging toseparate from one another. Bendall et al. “IGF and FGF cooperativelyestablish the regulatory stem cell niche of pluripotent human cells invitro.” Nature (2007) 448:1015-1021 (2007). In addition, these surfacestypically rely on ECM proteins from animal or human sources. See, e.g.,Amit M, et al., “Feeder layer- and serum-free culture of human embryonicstem cells,” Biol. Reprod. 70:837-845 (2004); Braam 5, et al.,“Recombinant vitronectin is a functionally defined substrate thatsupports human embryonic stem cell self renewal via αVβ5 integrin,” StemCells [Epub ahead of print, Jul. 17, 2008]; and Xu et al., supra.

As such, the art desires insoluble substrates with chemically definedsurfaces and culture conditions for pluripotent cells that support theirlong-term growth and maintenance.

Further, there is a great need for methods for differentiatingpluripotent cells on defined substrates and separating thedifferentiated cells from undifferentiated pluripotent cells.

BRIEF SUMMARY

In a first aspect, the present invention is summarized as an insolublesubstrate that presents a peptide that binds to a glycosaminoglycan(GAG). In one embodiment, the substrate has a chemically defined surfacethat presents a GAG-binding peptide that includes positively chargedamino acid residues or basic (i.e., hydrophilic) amino acid residuesseparated by one or two hydrophobic amino acid residues (i.e., XZX,wherein X can be independently lysine, arginine or histidine and Z canbe isoleucine, valine, leucine, phenalalanine, proline, methionine oralanine, and SEQ ID NO: 48, respectively), where the peptide occupies anarea between about 0.5% to about 100%, about 0.5% to about 50%, about 1%to about 5% or about 1% of the peptide-presenting surface. In oneembodiment, the peptide occupies an area that is at least 30% of thesurface. The peptide can contain a GAG-binding motif and can be asynthetic peptide or a GAG-binding peptide portion of a longerpolypeptide. A suitable peptide, without limitation, can range in lengthat least between about 3 and about 35 amino acids. A preferred peptidecan range in length between about 7 and about 18 amino acids. A fouramino acid GAG-binding peptide is described in the paper by Fromm,infra. The substrate having the chemically defined surface is suitablefor long-term culture of pluripotent cells.

In a second aspect, the present invention is summarized as a cellculture vessel that includes a chemically defined surface that presentsa peptide that includes positively charged amino acid residues or basic(i.e., hydrophilic) amino acid residues separated by one or twohydrophobic amino acid residues, where the peptide occupies an areabetween about 0.5% to about 25%, about 1% to about 5% or about 1% of thepeptide-presenting surface. The culture vessel can also include achemically defined medium that contains an effective amount of a kinaseinhibitor.

In a third aspect, the present invention is summarized as a cell culturemethod that includes the step of culturing pluripotent cells on asurface as defined above.

In a fourth aspect, the present invention is summarized as a method forseparating differentiated from undifferentiated cells that includes thestep of culturing pluripotent cells on a surface as defined above,inducing differentiation, and separating differentiated fromundifferentiated cells.

In a fifth aspect, the present invention is summarized as a compositionhaving a surface presenting GAG-binding peptides as defined above andpluripotent stem cells adhering to the surface, wherein the cellsdisplay a normal karyotype, pluripotent cell-specific markers, and anability to differentiate into all three germ layers after more thanthree months of culture on the surface.

In some embodiments, the chemically defined, peptide-presenting surfacecan include a self-assembled monolayer that includes one or morelong-chain alkanethiol (AT) having, e.g., a structure of X(CH₂)_(n)SH,where n is between about 3 and about 50. In a preferred embodiment, n isbetween about 11 and about 18.

In some embodiments, the peptide can be a GAG-binding peptide (GBP),such as the heparin-binding domain from vitronectin (GKKQRFRHRNRKG; SEQID NO:1), from fibronectin (GWQPPRARI; SEQ ID NO:2) or from bonesialoprotein (FHRRIKA; SEQ ID NO:3).

In some embodiments, the kinase inhibitor can be a Rho-associated kinase(ROCK) inhibitor, such as Y-27632[(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamidedihydrochloride], H-1152 [(S)-(+)-(2-methyl-5-isoquinolinyl)sulfonylhomopiperazine], and HA-100[1-(5-isoquinolinesulfonyl)piperazine hydrochloride].

The chemically defined surfaces can be used for growth and maintenanceof pluripotent cells. Even after long-term (i.e., >3 months) culture onthese surfaces, pluripotent cells retain a normal karyotype, pluripotentcell-specific markers characteristic of pluripotent cells and an abilityto differentiate into all three germ layers (e.g., endoderm, mesodermand ectoderm). In addition, these surfaces can display combinations ofadhesive ligands/epitopes with control over ligand/epitope density,location and composition. These surfaces also minimize the exposure ofpluripotent cells to potentially hazardous contaminants and/or animalproducts.

These and other features, objects and advantages of the presentinvention will become better understood from the description thatfollows. The description of preferred embodiments is not intended tolimit the invention or to cover all modifications, equivalents andalternatives. Reference should therefore be made to the claims hereinfor interpreting the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood and features, aspectsand advantages other than those set forth above will become apparentwhen consideration is given to the following detailed descriptionthereof. Such detailed description makes reference to the followingdrawings, wherein:

FIG. 1 shows the effects on pluripotent cell growth and maintenance ofvarious peptides spotted onto a peptide-AT array in a mixedself-assembling monolayer (SAM). FIG. 1A shows a representative arraypresenting bioactive peptides and surface densities, which are reportedas the percentage of peptide-ATs in a mixed SAM. Human ESCs (hESCs)bound to the surface in a peptide-specific and peptide-density dependentmanner. Over 6 days, cells proliferated to fill the array elements(FIGS. 1B-G; higher magnification image of cells on the array stainedfor Oct-4 and SSEA-4 and counterstained with DAPI). FIG. 1B shows hESCsgrown on a surface presenting a GBP derived from vitronectin(GKKQRFRHRNRKG; SEQ ID NO:1). FIG. 1C shows hESCs grown on a surfacepresenting a GBP derived from fibronectin (GWQPPRARI; SEQ ID NO:2). FIG.1D shows hESCs grown on a surface presenting a GBP derived from bonesialoprotein (FHRRIKA; SEQ ID NO:3). FIG. 1E shows hESCs grown on asurface presenting a FGF receptor binding peptide (GGGEVYVVAENQQGKSKA;SEQ ID NO:4) and an integrin-binding peptide (KGRGDS; SEQ ID NO:5). FIG.1F shows hESCs grown on a surface presenting the integrin-bindingpeptide (KGRGDS; SEQ ID NO:5) and another bioactive peptide derived fromfibronectin (KPHSRN; SEQ ID NO:6). FIG. 1G shows hESCs grown on asurface presenting a laminin-derived bioactive peptide (GSDPGYIGSR; SEQID NO:7).

FIG. 2 shows that GBPs support pluripotent cell self-renewal over atleast several passages. FIG. 2A shows that soluble heparin can abrogatehESC binding to surfaces coated with the heparin-binding peptideGKKQRFRHRNRKG (SEQ ID NO: 1), but not to surfaces coated with Matrigelor vitronectin. Percentages of cell binding represent the ratio of themean luminescence of cell lysates prepared from cells plated in thepresence of heparin versus those without heparin. The error barsindicate the standard deviation. FIG. 2B shows that hESCs cultured for 3passages on SAMs presenting GBPs or a combination of the vitronectin GBP(GKKQRFRHRNRKG; SEQ ID NO:1) and the integrin-binding peptide (KGRGDS;SEQ ID NO:5) maintained pluripotent cell-specific marker expression(Oct-4, SSEA-3, and SSEA-4; SSEA-1 served as a marker ofdifferentiation). Cells cultured on SAMs presenting the integrin-bindingRGD peptide (KGRGDS; SEQ ID NO:5) alone, however, had significantlylower levels of pluripotent cell-specific markers after 3 passages.Cells also grew much more slowly on RGD-presenting surfaces than onsurfaces presenting a GAG-binding peptide.

FIG. 3 shows that synthetic surfaces presenting the vitronectin GBP(GKKQRFRHRNRKG; SEQ ID NO:1) support pluripotency and karyotypestability of hESCs over 3 months of culture. FIG. 3A shows that hESCscultured on chemically defined surfaces for 3 months maintained highlevels of markers of pluripotency. Error bars represent an average from3 consecutive passages after passage 17. FIG. 3C shows that hESCs (H9hESCs) cultured on vitronectin GBP (GKKQRFRHRNRKG; SEQ ID NO:1) forabout 3 months were karyotypically normal as determined by standard Gbanding.

FIG. 4 shows a list of heparin-binding peptide sequences.

FIG. 5 shows growth characteristics of hESCs cultured on natural andsynthetic substrates.

While the present invention is susceptible to various modifications andalternative forms, exemplary embodiments thereof are shown by way ofexample in the drawings and are herein described in detail. It should beunderstood, however, that the description of exemplary embodiments isnot intended to limit the invention to the particular forms disclosed,but on the contrary, the intention is to cover all modifications,equivalents and alternatives falling within the spirit and scope of theinvention as defined by the appended claims.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

The present invention relates to the inventors' observation thatpluripotent cells have cell surface receptors that recognize and adhereto GBPs, such as the heparin-binding peptide from vitronectin (i.e.,GKKQRFRHRNRKG; SEQ ID NO:1). The inventors' therefore hypothesized thatGBPs can be displayed on a surface as a synthetic alternative to a hostof extracellular matrix proteins, such as those present in Matrigel®.

As described below, insoluble substrates with chemically definedsurfaces presenting GBPs supported both pluripotent cell attachment andself-renewal, as assessed by the presence of Oct-4 and SSEA-4 after 6days of culture. Although certain GBPs are known to promote celladhesion and spreading, they have not been previously shown to supportpluripotent cell self-renewal. See, e.g., McCarthy J, et al.,“RGD-independent cell adhesion to the carboxy-terminal heparin-bindingfragment of fibronectin involves heparin-dependent and -independentactivities,” J. Cell Biol. 110:777-787 (1990); and Vogel B, et al., “Anovel integrin specificity exemplified by binding of the alpha v beta 5integrin to the basic domain of the HIV Tat protein and vitronectin,” J.Cell Biol. 121:461-468 (1993), each of which is incorporated herein byreference as if set forth in its entirety.

The chemically defined, peptide-presenting surfaces described herein areuseful in a variety of contexts and applications. For example, thesurfaces can be used for maintaining pluripotent cells in anundifferentiated state. In addition, the surfaces can be used forexpanding a population of pluripotent, yet undifferentiated, cells. Thechemically defined, peptide-presenting surfaces are also useful forculturing pluripotent cells that are subsequently induced todifferentiate by, for example, adding one or more differentiation agentto the media. Differentiated cells derived from pluripotent cells can bemaintained on the chemically defined surfaces.

Cell types pass through various levels of potency duringdifferentiation, such as totipotency, pluripotency and multipotency. Ofparticular interest herein are pluripotent cells. As used herein, a“pluripotent cell” or “pluripotent cells” means a cell or population ofcells that can differentiate into all three germ layers (e.g., endoderm,mesoderm, and ectoderm). Pluripotent cells express a variety ofpluripotent cell-specific markers (e.g., Oct-4, SSEA-3, SSEA-4,Tra-1-60, or Tra-1-81, but not SSEA-1), have a cell morphologycharacteristic of undifferentiated cells (e.g., compact colony, highnucleus to cytoplasm ratio and prominent nucleolus) and form teratomaswhen introduced into an immunocompromised animal, such as a SCID mouse.See, e.g., Evans & Kaufman, supra. The teratomas typically contain cellsor tissues characteristic of all three germ layers. One can assess thesecharacteristics by using assays commonly used in the art. See, e.g.,Thomson J, et al., “Embryonic stem cell lines derived from humanblastocysts,” Science 282:1145-1147 (1998), incorporated herein byreference as if set forth in its entirety.

Pluripotent cells are capable of proliferating in cell culture anddifferentiating towards a variety of lineage-restricted cell populationsthat exhibit multipotent properties. Pluripotent cells have a higherpotency than multipotent cells, which are somatic cells that are moredifferentiated relative to pluripotent cells, but are not yet terminallydifferentiated.

Suitable pluripotent cells for use herein include ESCs and iPS cells,which preferably are from a primate, especially a human primate. As usedherein, “embryonic stem cells” or “ESCs” mean a pluripotent cell orpopulation of pluripotent cells derived from an inner cell mass of ablastocyst. See, Thomson et al., supra. These cells express at leastOct-4, SSEA-3, SSEA-4, TRA-1-60 or TRA-1-81, and appear as compactcolonies having a high nucleus to cytoplasm ratio and prominentnucleolus. ESCs are commercially available from sources such as WiCellResearch Institute (Madison, Wis.).

As used herein, “induced pluripotent stem cells” or “iPS cells” mean apluripotent cell or population of pluripotent cells that may vary withrespect to their differentiated somatic cell of origin, that may varywith respect to a specific set of potency-determining factors and thatmay vary with respect to culture conditions used to isolate them, butnonetheless are substantially genetically identical to their respectivedifferentiated somatic cell of origin and display characteristicssimilar to higher potency cells, such as ESCs, as described herein. See,e.g., Yu J, et al., “Induced pluripotent stem cell lines derived fromhuman somatic cells,” Science 318:1917-1920 (2007), incorporated hereinby reference as if set forth in its entirety.

iPS cells exhibit morphological properties (e.g., round shape, largenucleoli and scant cytoplasm) and growth properties (e.g., doubling timeof about seventeen to eighteen hours) akin to ESCs. In addition, iPScells express pluripotent cell-specific markers (e.g., Oct-4, SSEA-3,SSEA-4, Tra-1-60 or Tra-1-81, but not SSEA-1). iPS cells, however, arenot immediately derived from embryos. As used herein, “not immediatelyderived from embryos” means that the starting cell type for producingiPS cells is a non-pluripotent cell, such as a multipotent cell orterminally differentiated cell, such as somatic cells obtained from apost-natal individual.

Other types of pluripotent cells suitable for use herein include, butare not limited to, cells from somatic cell nuclear transfer (see, e.g.,Wilmut I, et al., “Viable offspring derived from fetal and adultmammalian cells,” Nature 385:810-813 (1997)) or cells from fusion ofsomatic cells with ESCs (see, e.g., Cowan C, et al., “Nuclearreprogramming of somatic cells after fusion with human embryonic stemcells,” Science 309:1369-1373 (2005); and Yu et al., “Human embryonicstem cells reprogram myeloid precursors following cell-cell fusion,”Stem Cells 24:168-176 (2006)).

Regardless of the pluripotent cell used, the chemically defined surfacesdescribed herein can be constructed according to known methods. Forexample, one can use contact spotting of peptides ontoglyoxylyl-functionalized glass slides (see, e.g., Falsey J, et al.,“Peptide and small molecule microarray for high throughput cell adhesionand functional assays,” Bioconjug. Chem. 12, 346-353 (2001)); contactprinting of peptides onto acrylamide-coated glass slides; and spottingcombinations of peptides onto a glass slide followed by in situpolymerization (see, e.g., Anderson et al., Nanoliter-scale synthesis ofarrayed biomaterials and application to human embryonic stem cells, Nat.Biotechnol. 22:863 (2004). In addition, one can use streptavidin-coatedplates treated with a biotinylated peptide of interest or evenpolyacrylamide gels cross-linked to a peptide of interest. See, e.g.,Klein et al., Cell adhesion, cellular tension, and cell cycle control,Meth. Enzymol. 426:155 (2007). Water-insoluble synthetic or naturalhydrogels are also contemplated as providing a suitablepeptide-presenting surface.

As used herein, a “glycosaminoglycan” (GAG) is a polysaccharide composedof repeating disaccharide units and amino sugars. Glycosaminoglycans arenegatively charged and can be linked to proteins to form proteoglycans.Examples of glycosaminoglycans include chondroitin sulfate, dermatansulfate, heparin, heparan sulfate, hyaluronate, and keratan sulfate.

Preferably, one spots ATs onto an inert background to formself-assembling monolayers (SAMs). See, e.g., Derda et al., supra; DerdaR, et al., “Solid-phase synthesis of alkanethiols for the preparation ofself-assembled monolayers,” Langmuir 23:11164-11167 (2007); and HousemanB & Mrksich M, “Efficient solid-phase synthesis of peptide-substitutedalkanethiols for the preparation of substrates that support the adhesionof cells,” J. Org. Chem. 63:7552-7555 (1998), each of which isincorporated herein by reference as if set forth in its entirety. Forexample, a background can be formed of perfluoro-AT, which can be bothcytophobic (i.e., repel cells) and solvophobic (i.e., repel solvents).First, a gold-coated surface can be coated with a perfluoro-ATmonolayer, either leaving areas (i.e., holes) in the monolayer for theelements of an array or creating areas in the monolayer for the elementsof the array in a subsequent step. Then, ATs coupled to a peptide, suchas an GBP as described herein, can be attached to the substrate in theholes to complete the monolayer and to present ligands for pluripotentcell attachment to the surface. Surface density of the GBPs can becontrolled using mixed SAMs of peptide-ATs and non-adhesiveglucamine-ATs. Methods for synthesizing the AT species for both regionson the chemically defined surface array are provided in the examplesbelow. For long-term culture on gold-coated coverslips (as opposed toarray elements), a mixed monolayer of peptide-AT and glucamine-AT (5%peptide-AT) was formed. After 24 hours, the surfaces were washed withethanol and cells were plated on the surfaces.

Each AT species may be thought of as having three important regions ormoieties. One region is at the basal end, which is an attachment moietyintended to attach the monolayer species to the surface. The attachmentmoiety is typically a thiol group, which attaches to the gold substrate.Other attachment groups can attach to other substrates. Another regionis the intermediate region, which is a spacer moiety, such as an alkaneof between about 3 and about 50 carbons in length, and preferablybetween about 11 and about 18 carbons in length, as described elsewherein this application. Other simple organic groups can be used for thespacer as long as the resulting species are capable of self-assembly ina monolayer. Lastly, the active group at the end of the monolayerspecies is the ligand, which can be a group intended to be cytophobic orcytophilic. Cytophilic ligands suitable for use herein include, but arenot limited to, a peptide, especially a peptide having basic amino acidresidues separated by one or two hydrophobic amino acid residues, likethe vitronectin GBP (SEQ ID NO:1), fibronectin GBP (SEQ ID NO:2) andbone sialoprotein GBP (SEQ ID NO:3). As shown in FIG. 5, not all GBPsequences possess basic amino acid residues separated by one or twohydrophobic amino acid residues. Indeed, the inventors predict that if apeptide binds a gycosaminoglycan, it is capable of self renewingpluripotent stem cells.

Basic (i.e., hydrophilic) amino acids are polar and positively chargedat pH values below their pKa's. Examples of basic amino acids includelysine, histidine and arginine.

The hydropathy index of an amino acid is a number representing thehydrophobic or hydrophilic properties of its side-chain. See, Kyte J &Doolittle R, “A simple method for displaying the hydropathic characterof a protein,” J. Mol. Biol. 157:105-132 (1982). The larger the numberis, the more hydrophobic the amino acid. The most hydrophobic aminoacids are isoleucine (4.5) and valine (4.2); whereas the mosthydrophilic ones are arginine (−4.5) and lysine (−3.9). Hydropathy isimportant in protein structure, as hydrophobic amino acids tend to beinternal (with regard to the protein's three-dimensional shape), whilehydrophilic amino acids are more commonly found towards the protein'ssurface.

TABLE 1 Hydropathy index for the twenty natural amino acids (Kyte &Doolittle). A R N D C Q E G H I L K M F P S T W Y V 1.8 −4.5 −3.5 −3.52.5 −3.5 −3.5 −0.4 −3.2 4.5 3.8 −3.9 1.9 2.8 −1.6 −0.8 −0.7 −0.9 −1.34.2

TABLE 2 Amino acids sorted by increasing hydropathy index. R K N D Q E HP Y W S T G A M C F L V I −4.5 −3.9 −3.5 −3.5 −3.5 −3.5 −3.2 −1.6 −1.3−0.9 −0.8 −0.7 −0.4 1.8 1.9 2.5 2.8 3.8 4.2 4.5

An advantage of using ATs is that they form reproducible SAMs andchemically defined surfaces. This attribute means that the surfacescreated are chemically defined surfaces that will vary only because ofthe peptide or additional ligand(s) presented on the surface and notbecause of other bulk properties of the surface, such as topology.Another advantage of using ATs is that the peptide or additionalligand(s) can be engineered to be presented to the pluripotent cells indefined areas of the surface and that other areas of the surface (i.e.,background areas) can be engineered to resist both solvents and cellpresence. In contrast to the surfaces previously constructed for cultureof cells, the components of the chemically defined surfaces describedherein are fully characterized with known quantities of all ingredients.

Long-term culture of pluripotent cells on the chemically definedsurfaces described herein typically will begin by chemically,enzymatically or mechanically dissociating confluent pluripotent cellsfrom a surface, such as Matrigel® or MEFs, into clumps/aggregates oreven single cells. In some cases, the surface will be one of thechemically defined surfaces described herein, e.g. when platingconfluent cells onto a fresh chemically defined surface.

The clumps or aggregates or single cells then can be plated onto achemically defined surface as described herein in a protein-free basalmedium such as Dulbecco's Modified Eagle's Medium (DMEM)/F12 or mTeSR oreven phosphate-buffered saline (PBS) or Hank's Balanced Salt Solution(HBSS) to minimize any non-specific adsorption of proteins from themedium. After about 1 hour, the medium can be replaced with a definedculture medium such as, e.g., mTeSR™1 supplemented with a kinaseinhibitor.

As used herein, a “chemically defined medium,” “defined culture medium”or “defined medium” means that the medium has known quantities of allingredients. Typically, serum that is normally added to culture mediumfor cell culture is replaced by known quantities of serum components,such as, e.g., albumin, insulin, transferrin and possibly specificgrowth factors (i.e., basis fibroblast growth factor, transforminggrowth factor or platelet-derived growth factor). Defined medium (DM) istherefore serum-free. As used herein, “serum-free” means that a mediumdoes not contain serum or serum replacement, or that it containsessentially no serum or serum replacement. As used herein, “essentially”means a de minimus or reduced amount (i.e., less than 5%) of acomponent, such as serum, may be present.

An example of a DM suitable for use herein is TeSR™. The fullconstituents and methods of use of TeSR™ are described in Ludwig et al.See, Ludwig T, et al., “Feeder-independent culture of human embryonicstem cells,” Nat. Methods 3:637-646 (2006); and Ludwig T, et al.,“Derivation of human embryonic stem cells in defined conditions,” Nat.Biotechnol. 24:185-187 (2006), each of which is incorporated herein byreference as if set forth in its entirety. Other DM formulationssuitable for use herein include, e.g., mTeSR™ (StemCell Technologies;Vancouver, British Columbia, Canada), X-Vivo (BioWhittaker,Walkersville, Md.) and StemPro® (Invitrogen; Carlsbad, Calif.).

Kinase inhibitors, such as ROCK inhibitors, are known to protect singlecells and small aggregates of cells. See, e.g., US Patent ApplicationPublication No. 2008/0171385, incorporated herein by reference as if setforth in its entirety; and Watanabe K, et al., “A ROCK inhibitor permitssurvival of dissociated human embryonic stem cells,” Nat. Biotechnol.25:681-686 (2007). ROCK inhibitors are shown below to significantlyincrease pluripotent cell survival on chemically defined surfaces. ROCKinhibitors suitable for use herein include, but are not limited to,(S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]homopiperazinedihydrochloride (informal name: H-1152),1-(5-isoquinolinesulfonyl)piperazine hydrochloride (informal name:HA-100), 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (informal name:H-7), 1-(5-isoquinolinesulfonyl)-3-methylpiperazine (informal name: isoH-7), N-2-(methylamino) ethyl-5-isoquinoline-sulfonamide dihydrochloride(informal name: H-8), N-(2-aminoethyl)-5-isoquinolinesulphonamidedihydrochloride (informal name: H-9),N-[2-p-bromo-cinnamylamino)ethyl]-5-isoquinolinesulfonamidedihydrochloride (informal name: H-89),N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (informalname: HA-1004), 1-(5-isoquinolinesulfonyl) homopiperazinedihydrochloride (informal name: HA-1077),(S)-(+)-2-Methyl-4-glycyl-1-(4-methylisoquinolinyl-5-sulfonyl)homopiperazinedihydrochloride (informal name: glycyl H-1152) and(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamidedihydrochloride (informal name: Y-27632). The kinase inhibitor can beprovided at a concentration sufficiently high that the cells survive andremain attached to the surface. An inhibitor concentration between about3 μM to about 10 μM can be suitable. At lower concentrations, or when noROCK inhibitor is provided, undifferentiated cells typically detach,while differentiated cells remain attached to the defined surface.

The inventors have exploited the observation that undifferentiated butnot differentiated cells require ROCK inhibitor for attachment to thechemically defined surfaces to separate differentiated fromundifferentiated cells. For example, pluripotent or multipotent cellscan be maintained on the chemically defined surfaces in the presence ofROCK inhibitor. At a desired time, the cells can be induced todifferentiate on the chemically defined surfaces by, for example, addingone or more differentiation agent to the cell culture medium.Alternatively, pluripotent and non-pluripotent cells can be plated ontothe chemically defined surfaces directly. To separate differentiatedfrom undifferentiated cells, ROCK inhibitor is removed from the culturemedia, such that undifferentiated, but not differentiated, cells detachfrom the surface leaving behind a population of attached, differentiatedcells.

During culture on the chemically defined surface, conventional cellculture conditions can be used. For example, the temperature can varybetween about 36° C. to about 37.5° C. Likewise, the CO₂ concentrationcan, and will, vary between about 2% to about 10% depending on themedium and bicarbonate concentration. For example, the cell cultureconditions can be 37° C. and 5% CO₂ in a humidified chamber. Thepluripotent cells can be cultured to confluence (typically about 5 daysto about 6 days), at which time, they can be passaged by methods knownin the art (i.e., by chemical, enzymatic or mechanical means).

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar to or equivalent to those described herein can be usedin the practice or testing of the present invention, the preferredmethods and materials are described herein.

In describing the embodiments and claiming the invention, the followingterminology will be used in accordance with the definitions set outbelow.

As used herein, “about” means within 5% of a stated concentration range,density, temperature, or time frame.

As used herein, “homologous” refers those polypeptides sharing at least90% or at least 95% sequence identity to a given GBP (e.g., SEQ IDNOS:1-3) that result in binding by pluripotent cells via the cellsurface. For example, a polypeptide that is at least 90% or at least 95%identical to the GBPs discussed herein is expected to be a constituentof a complex between the peptide and a molecule on the exterior surfaceof a pluripotent cell. One of ordinary skill in the art understands thatmodifications to the polypeptide can include substitutions, insertions(e.g., adding no more than ten amino acid) and deletions (e.g., deletingno more than ten amino acids). These modifications can be introducedinto the polypeptides discussed herein without abolishing structure andultimately, function. Polypeptides containing such modifications can beused in the methods described herein.

In addition, it is well known in the art that amino acids within thesame conservative group can typically substitute for one another withoutsubstantially affecting the function of a protein. For the purpose ofthe present invention, such conservative groups are set forth in Table 3and are based on shared properties.

TABLE 3 Amino Acid Conservative Substitutions. Original ResidueConservative Substitution Ala (A) Val, Leu, Ile Arg (R) Lys, Gln, AsnAsn (N) Gln, His, Lys, Arg Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E)Asp His (H) Asn, Gln, Lys, Arg Ile (I) Leu, Val, Met, Ala, Phe Leu (L)Ile, Val, Met, Ala, Phe Lys (K) Arg, Gln, Asn Met (M) Leu, Phe, Ile Phe(F) Leu, Val, Ile, Ala Pro (P) Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr,Phe Tyr (Y) Trp, Phe, Thr, Ser Val (V) Ile, Leu, Met, Phe, Ala

The gene and protein sequences for vitronectin, fibronectin and bonesialoprotein are known and characterized, see, e.g., GeneID numbers7448, 2335, and 3381, respectively. All of the sequences defined by theabove-noted GeneID numbers are incorporated by reference here in theirentirety. Also, information about other GAG-binding molecules that canbe used to support pluripotent stem cell self renewal is found, forexample, in Fromm, J. R., et al., Pattern and Spacing of Basic AminoAcids in the Heparin Binding Sites, Arch. Biochem. Biophys. 343:92(1997); which is incorporated by reference here in its entirety.

Each mentioned publication is incorporated by reference as if set forthherein in its entirety.

The invention will be more fully understood upon consideration of thefollowing non-limiting Examples.

EXAMPLES Example 1 Culture and Self-Renewal of hESCs and iPS Cells onChemically Defined Surfaces Presenting GBPs

Methods:

Cell culture: H1, H7, H9, H13, or H14 hESCs (WiCell Research Institute)and iPS cells (DF19-9 and IMR90-derived iPS cells) were maintained onMatrigel®-coated plates (Matrigel® obtained from BD Biosciences;Franklin Lakes, N.J.) using mTeSR™1 medium. The cells were maintained at37° C. and 5% CO₂ and manually passaged every 5-6 days after treatingwith 2 mg/ml Dispase® (Gibco; Rockville, Md.) for 5-6 minutes.

hESCs were passaged to SAMs of peptide-AT conjugates on gold in mTeSR™1optionally supplemented with 10 ng/ml heregulin-β1 (Peprotech; RockyHill, N.J.) and 5 μM Y-27632 (Calbiochem; San Diego, Calif.). Theheregulin evidenced modest improvement of initial cell survival. Somesurfaces presented peptide-AT conjugates of only vitronectin GBP (SEQ IDNO:1), fibronectin GBP (SEQ ID NO:2) or bone sialoprotein GBP (SEQ IDNO:3) Alternatively, some surfaces presented peptide-AT conjugates ofonly the integrin-binding peptide (SEQ ID NO:5), another bioactivepeptide derived from fibronectin (SEQ ID NO:6), the FGF receptor bindingpeptide (SEQ ID NO:4), the laminin-derived bioactive peptide (SEQ IDNO:7), ADSQLIHGGLRS (SEQ ID NO: 8) or MHRMPSFLPTTL (SEQ ID NO: 9).Furthermore, some surfaces presented separate peptide-AT conjugates ofthe integrin-binding peptide (SEQ ID NO:5) and FGF receptor bindingpeptide (SEQ ID NO:4); the integrin-binding peptide (SEQ ID NO:5) andvitronectin GBP (SEQ ID NO:1); the integrin-binding peptide (SEQ IDNO:5) and bioactive peptide derived from fibronectin (SEQ ID NO:6); andthe vitronectin GBP (SEQ ID NO:1) and MRHMPSFLPTTL (SEQ ID NO: 9). Thedensity of the peptides on the surface varied from 0.5% to 25% (see,FIG. 1).

Moreover, some hESCs were passaged to streptavidin-coated plates treatedwith biotinylated vitronectin GBP (SEQ ID NO:1) or polyacrylamide gelscross-linked to vitronectin GBP (SEQ ID NO:1).

Regardless of the nature of the chemically defined surface or peptide(s)attached thereto, about 5×10⁴ cells/ml were manually passaged to a freshchemically defined surface every 5-7 days (i.e., at confluency) aftertreatment with an Enzyme-Free Cell Dissociation Buffer (Sigma; St.Louis, Mo.; a phosphate-buffered saline (PBS)+ethylenediaminetetraacetic acid (EDTA) 0.02% wt/vol) for 10-15 minutes. After threepassages (about 21 days), the cells were evaluated for pluripotentcell-specific markers by flow cytometry.

Cell Adhesion: To determine the effect of a soluble GAG on hESC bindingto GBPs, hESCs were plated onto Matrigel-coated surfaces,vitronectin-coated surfaces, or surfaces presenting the heparin-bindingpeptide GKKQRFRHRNRKG (SEQ ID NO: 1) in the presence or absence ofsoluble heparin (0.5 mg/mL). After 1 hour, the surfaces were washed andthe cells lysed. The cell lysates were used to determine approximatecell numbers using Cell Titer Glo (Promega). The ratio of the meanluminescence of the cell lysates of cells plated in the presence ofheparin versus those without heparin was expressed as percent cellbinding for each surface.

To determine if glycosaminoglycans (GAGs) are required for GBP binding,human ESCs (H9) cultured on Matrigel were dissociated using anenzyme-free, Hanks'-based cell dissociation buffer (Sigma) for 10-15minutes. Cells were resuspended in DMEM/F12 (Gibco), or DMEM/F12supplemented with 2 units/mL chondroitinase ABC (Sigma), or 500 ug/mLheparin (Sigma). Cells treated with the GAG-degrading enzymes wereincubated for 1 hour in suspension at 37° C. Cell suspensions wereseeded onto Matrigel-coated surfaces, recombinant vitronectin-coatedsurfaces (10 μg/mL, R&D Systems), or SAMs presenting the peptideGKKQRFRHRNRKG (SEQ ID NO: 1) at a 5% surface density. After 1 hour,surfaces were washed 3 times with PBS and the cells were lysed withM-PER buffer (Thermo Fisher Scientific, Inc., Rockford, Ill.). The celllysate was mixed with CellTiter-Glo (Promega) to determine the number ofviable cells in culture based on the presence of ATP. The luminescencewas measured on 20/20^(n) luminometer (Turner Biosystems, Inc.,Sunnyvale Calif.).

Cell Growth: hESCs (H9) were cultured on Matrigel, vitronectin,polylysine, GKKQRFRHRNRKG (SEQ ID NO: 1), or KGRGDS (SEQ ID NO: 5) overtwo passages in mTeSR media supplemented with 5 μm ROCK inhibitor. Cellcounts were calculated at each time point using Cell Counting Kit-8(Dojindo Molecular Technologies, Inc., Rockville, Md.).

hESC Differentiation: After culture for more than 3 months, hESCs wereallowed to form embryoid bodies (EBs) in a suspension culture, whichwere formed in poly(2-hydroxyethyl methacrylate)-coated flasks (GreinerBio-One; Monroe, N.C.), and cultured in a medium of Iscove's ModifiedDulbecco's Medium (Gibco), 15% fetal bovine serum (FBS; Gibco), 1%non-essential amino acids (Gibco) and 0.1 mM β-mercaptoethanol (Gibco).

Microscopy and Immunostaining: Images were collected with a Hamamatsu(Bridgewater, N.J.) Digital Camera mounted onto an Olympus IX81Microscope. Primary antibodies used were as follows: Oct-4 (1:400; R&DSystems; Minneapolis, Minn.), SSEA-4 (1:400; Santa Cruz Biotechnology;Santa Cruz, Calif.), β-III tubulin (1:3000; R&D Systems), nestin(1:3000), α-fetoprotein (1:250; Sigma), FoxA2 (1:100; R&D Systems),α-smooth muscle actin (1:1000; Sigma), and fatty acid binding protein 4(1:250; R&D Systems). Cells were fixed with PBS containing 4%formaldehyde and 0.15% picric acid for 20 minutes at 37° C. and thenpermeabilized and blocked with PBS containing 0.1% Triton X-100 and 0.1%bovine serum albumin (BSA). All antibodies were incubated in blockingbuffer overnight at 4° C., except for the antibodies against β-IIItubulin, nestin and α-smooth muscle actin, which were incubated for 1hour at room temperature. Secondary staining was performed with AlexaFluor® 488- and/or 594-conjugated antibodies (1:1000; Invitrogen)diluted in blocking buffer and incubated for 1 hour at room temperature.Cells were counterstained with 4′,6-diamidino-2-phenylindole, dilactate(DAPI; Invitrogen). Image overlays were generated using ImageJ (ImageJis a public domain Java image processing program available on the WorldWide Web). Peptide array mosaics were generated using the AnalySISAcquisition Software (Olympus).

Flow Cytometry: hESCs were dissociated with 0.05% trypsin-EDTA with 2%chicken serum (Gibco). Cell surface marker staining was performed in PBScontaining 2% BSA (wt/vol) at 4° C. for 30 minutes with directlyconjugated antibodies against alkaline phosphatase (R&D Systems), Tra1-60 (BD Biosciences), Tra 1-81 (BD Biosciences), SSEA-4 (BDBiosciences), SSEA-3 (BD Biosciences), SSEA-1 (R&D Systems) followed bya 30 minute fixation with 2% formaldehyde/PBS at room temperature.

For intracellular marker staining, hESCs were fixed with 2%formaldehyde/PBS at room temperature for 30 minutes. For Oct-4 staining,cells were permeabilized with saponin permeabilization buffer (SPB; 0.1%saponin, 0.1% BSA wt/vol in PBS) for 30 minutes at room temperaturefollowed by incubation with an Oct-4 PE-conjugated antibody overnight.Cells were then washed 2 times with SPB before analysis.

For Sox-2 staining, cells were permeabilized with 90% ice-cold methanol,washed with SPB, incubated with Sox-2 Alexa-Fluor® conjugated antibodyfor 1 hour at 4° C., and then washed 2 times with SPB.

Flow cytometry data was obtained using a FACSCalibur™ (BD Biosciences)and analyzed using FlowJo Software (Tree Star, Inc.; Ashland, Oreg.).The percentage of positive cells was established by comparingexperimental cells to partially differentiated hESCs. Gating forpositive and negative populations was established by analyzing thebimodal peaks of partially differentiated hESCs.

G-banded Karyotyping: Human ES cells were harvested as follows. Ethidiumbromide (0.001% final concentration; ThermoFisher Scientific,http://www.thermofisher.com, Waltham, Mass.) was added directly toactively dividing cultures (day 3 or 4 after passage) that were thenincubated for 40 minutes in a 37° C. incubator with 5% CO₂. Colcemid(200 ng/ml final concentration; Invitrogen) was added to the cultures,and they were returned to the incubator for an additional 30 minutes.Cells were disassociated with 0.05% trypsin-EDTA (Invitrogen),centrifuged, resuspended in 5 ml of 0.075M KCl hypotonic (Invitrogen),and incubated in a 37° C. waterbath for 18-25 minutes. Cell suspensionswere pre-fixed for five minutes at room temperature with 20 drops of 3:1methanol to acetic acid fixative (low water methanol, certified ACS plusacetic acid; ThermoFisher Scientific). Following centrifugation (200×gfor 5 min), the pre-fixation solution was removed, replaced withfixative, and incubated at room temperature for 30 min. Fixative wasreplaced at least 2 more times. Fixed cell suspensions were dropped ontoglass slides under controlled temperature and humidity conditions (25°C./33% humidity) in a CDS-5 Cytogenetics Drying Chamber (Thermotron,Holland, Mich., http://www.thermotron.com). These preparations wereheated for 1 hour at 90° C. on a ThermoBrite StatSpin (Abbott) to “age”the metaphase spreads for G-banding and allowed to cool to roomtemperature. Slides were dipped in 1× trypsin-EDTA (0.05%, diluted inHBSS (Invitrogen)) for 25-30 seconds followed by brief washes in a FBSsolution (2% (v/v), Invitrogen, diluted in HBSS) and MilliQ water(Millipore). Chromosomes were stained for 90 seconds with Leishman'sstain (0.2% (w/v), Sigma, dissolved in methanol (Fisher)) diluted 1:4 inGurr buffer (Invitrogen), followed by two brief washes in MilliQ water(Millipore). The slides were dried on a 50° C. hotplate for 15 min andcoverslipped with Cytoseal-60 mounting media (Richard-Allan Scientific,Kalamazoo, Mich., http://www.rallansci.com). G-Band analysis wasperformed by random selection of at least 20 metaphases. Chromosomes ineach selected metaphase cell were counted to establish modal chromosomenumber, a minimum of 8 were analyzed microscopically, and at least 4 ofthese were karyogrammed. Metaphase images were captured and analyzedwith the Applied Spectral Imaging (ASI) acquisition and B and Viewsoftware (Vista, Calif., http://www.spectral-imaging.com) with anOlympus BX41 microscope (Olympus, Center Valley, Pa.,http://www.olympusamerica.com).

Defined Surface Fabrication: Chromium (1 nm) and then gold (25 nm) wereevaporated onto piranha solution-cleaned glass coverslips (Corning No1½, 23 mm squares) using a thermal evaporator (Denton Vacuum;Moorestown, N.J.). Substrates were immediately immersed into a 1 mMsolution of fluoro-AT in absolute ethanol. After 24 hours, substrateswere thoroughly rinsed with ethanol and dried under a stream ofnitrogen. Coverslips with fluoro-AT SAM were irradiated with UV-light (1kW—Hg—Xe Research Arc Lamp; Spectra-Physics; Stratford, Conn.) through aquartz photomask (array of 500 μm or 750 μm squares, 0.067quartz-chromium mask (Photo Sciences, Torrance, Calif.)) for 1 hour.Irradiated samples were rinsed thoroughly using several repetitivewashes with absolute ethanol and distilled water and dried under astream of nitrogen.

Spotting of AT solutions onto the bare gold areas was performed within 2hours of the photolithography. Spotting was performed manually using aP2-Pipetman (Gilson; Middleton, Wis.) in a humidity chamber. Spottedarrays were stored in the humidity chamber for 12 hours and thoroughlywashed using repeated washes with ethanol and water. Rapid flow duringwashing was used to prevent cross-contamination of array spots.

Alternatively, glass slides coated with 250 Å Gold and 10 Å Chromium, 22mm square, 0.16 mm thick) were purchased from EMF Corporation. Arrayswere prepared as previously described. Derda, R. et al., “Definedsubstrates for human embryonic stem cell growth identified from surfacearrays,” ACS Chem. Biol. 2, 347-355 (2007), incorporated herein byreference as if set forth in its entirety. When larger areas ofpeptide-AT SAMs were needed, whole chips presenting the same SAM werefabricated by sandwiching solutions of peptide-AT/glucamine-AT betweentwo gold coated slides. SAMs were allowed to form in humidity chambersfor 24 h before use.

Peptide-ATs: Peptides were synthesized on a Pioneer™ Peptide SynthesisSystem (Applied Biosystems; Foster City, Calif.) using standard Fmocchemistry on Rink Amide AM Resin (Novabiochem; loading: 0.56 mmol/g).Peptide-AT conjugates were prepared similarly to Houseman & Mrksich,supra. Briefly, resin containing protected peptide with a freeN-terminus was swollen in dry THF, 5-fold excess of each of the compound1, HOBt and 1,3-diisopropylcarbodiimide (DIC) was added to the resinsuspension in THF. The resin was incubated for 12 hours and another3-fold excess of DIC and HOBt was added. After 3 hours, the resin wastested with the Kaiser Test (see, Kaiser E, et al., “Color test fordetection of free terminal amino groups in solid-phase synthesis ofpeptides,” Anal. Biochem. 34:595-598 (1970)), washed with DMF anddichloromethane and dried in vacuo. After cleavage withTFA/DIC/EDT/H₂O/phenol (36:1:1:1:1) for 2 hours and ether precipitation,conjugates were purified by preparative HPLC. Gradient used (percentageof mobile phase A): 100→0% 20 min, 0% 3 min. 0→100% 3 minutes. Peaks atretention time around 17 minutes were collected. Each purified samplewas analyzed by LCMS and H NMR. Note: The presence of triplet of1,1,1-triples δ 2.49 (t[111t], 2H, J=7.1 Hz, J_(HD)=1.0 Hz) in H NMR atAT-peptides in CD₃OD is indicative of free thiol functionality. It isthe signal of methylene hydrogens next to the free thiol functionality(7 NZ, coupling to neighboring methylene, 1 Hz coupling to deuterium onfree thiol).

Results:

The inventors examined whether GBPs, such as SEQ ID NO:1, alone or incombination with other adhesion peptides could support pluripotent cellattachment, growth, and maintenance using alternative methods ofpresentation. The chemically defined surfaces described herein showedexcellent attachment, self-renewal, and colony spreading, but required acertain degree of chemical expertise to produce.

hESCs (H1 and H9) propagated in mTeSR media supplemented with Y-27632,an inhibitor of Rho-associated coiled-coiled kinase (ROCK), on surfacespresenting GBPs for 6 days maintained high levels of Oct-4 and SSEA-4expression (FIG. 1). Cells treated with a glycosaminoglycan-degradingchondroitinase ABC enzyme maintained their ability to adhere to Matrigeland vitronectin but exhibited decreased adhesion to the syntheticsurfaces presenting GKKQRFRHRNKG (SEQ ID NO: 1). Soluble heparin, whichcompeted with cell surface GAGs for binding to the surface, alsoinhibited adhesion (FIG. 2A).

A variety of peptidic surfaces supported hESC attachment, growth, andmaintenance. Interestingly, peptide surfaces presenting GBP, likevitronectin GBP, best supported both cell attachment and self-renewal,as determined by the presence of Oct-4 and SSEA-4 after 6 days. Notably,while a combination of RGD peptides and vitronectin GBP supported cellself-renewal, integrin binding through RGD peptides was not necessaryfor self-renewal (see, FIG. 2).

RGD-presenting surfaces are inferior substrata for pluripotent stem cellpropagation. Cells cultured on GBPs displayed a high nucleus tocytoplasm ratio, maintained high levels of markers of pluripotency (FIG.2B), and grew in the form of tightly packed colonies characteristic ofundifferentiated hES cells. In contrast, cell populations grown onRGD-presenting surfaces grew in the form of heterogeneous mixtures ofcolonies and individual cells, and fewer cells in the populationdisplayed markers of pluripotency (FIG. 2B). Consistently, cellscultured on RGD-presenting surfaces exhibited a variety of morphologiesand differentiated into all three embryonic germ layers. Thus,attachment to RGD peptides alone was insufficient to supportself-renewal.

After 3 months of continuous passaging on defined surfaces presentingSEQ ID NO:1, cells were evaluated for pluripotent cell-specific markerssuch as Oct-4 and SSEA-4 (see, FIG. 3B), NANOG and SOX2, which weremaintained at high levels. Importantly, cells cultured on GBP-presentingsurfaces formed homogenous populations of undifferentiated pluripotentcells (FIG. 3A) that grew in densely packed colonies. In contrast, cellscultured on Matrigel® formed heterogeneous cell populations. While geneexpression levels of markers associated with pluripotency werecomparable between cells grown on Matrigel and cells grown onGBP-presenting surfaces, cells grown on Matrigel lost cell surfaceexpression of certain pluripotency markers (FIG. 3A). In contrast, cellsgrown on GBP-presenting surfaces maintained cell-surface expression ofthese markers. Furthermore, cells cultured on GBP-presenting surfacesremained karyotypically normal over the course of the experiment (see,FIG. 3B).

GBP-presenting surfaces are superior to polylysine-coated surfaces inpromoting cell divisions. The growth characteristics of pluripotent stemcells cultured on standard substrata were compared to those of cellscultured on surfaces presenting synthetic peptides. Growth curves weregenerated for hES cells (H9 and H13) and vector-free iPS cells (DF19-9)cultured on various surfaces over two passages. Matrigel-coated surfacesexhibited a slightly higher plating efficiency after 24 hours comparedto vitronectin- and GKKQRFRHRNKG (SEQ ID NO: 1)-coated surfaces, whichwere equivalent. Surfaces presenting the synthetic peptide KGRGDS (SEQID NO: 5) had the lowest plating efficiency (FIG. 5). Increased cellnumbers were observed after each passage for cells cultured onMatrigel-coated, vitronectin-coated, and GKKQRFRHRNKG (SEQ ID NO:1)-presenting surfaces (FIG. 5).

Cells cultured on synthetic surfaces presenting the heparin-bindingpeptide GKKQRFRHRNKG (SEQ ID NO: 1) or a combination of GKKQRFRHRNKG(SEQ ID NO: 1) and KGRGDS (SEQ ID NO: 5) underwent a similar number ofcell divisions as cells cultured on Matrigel, as indicated byfluorescent cell staining. In contrast, cells cultured onvitronectin-coated surfaces and synthetic surfaces presenting KGRGDS(SEQ ID NO: 5) underwent fewer cell divisions. Importantly, GKKQRFRHRNKG(SEQ ID NO: 1)-presenting surfaces are superior to polylysine-coatedsurfaces in promoting cell divisions.

Other chemically defined surfaces in addition to SAMs were also suitablefor culture of pluripotent cells. For example, streptavidin-coatedplates treated with biotinylated SEQ ID NO:1 supported cell adhesion andgrowth, although not to the same extent as when SEQ ID NO:1 waspresented on SAMs (data not shown). Interestingly, colony spreading waslower than on SAMs but ROCK inhibitor can be omitted without observableincrease in cell death, suggesting that other peptide-presentingscaffolds might not need ROCK inhibitor in the culture medium. Likewise,CGKKQRFRHRNRKG (SEQ ID NO: 47) conjugated to polyacrylamide gels orglass coverslips supported cell adhesion and growth comparable to SAMs.These results demonstrate that a variety of substrates that displayGAG-binding peptides can sustain pluripotent cell adhesion and growth.

Pluripotent cells cultured on the chemically defined, peptide-presentingsurfaces retained not only their ability to self-renew, but also theirability to differentiate into cells from the three germ layers. Afterculture on chemically defined surfaces presenting SEQ ID NO:1 for 3months, the cells subsequently formed EBs in a suspension culture. After2 weeks in the suspension culture, a heterogeneous population of cellswas stained for markers of all 3 embryonic germ layers, and stainedpositive for β-III tubulin and nestin, indicating derivatives of theectoderm. In addition, some cells stained positive for fatty acidbinding protein 4 and α-smooth muscle actin, indicating derivatives ofthe mesoderm. Finally, some cells stained positive for α-fetoprotein andFoxA2, indicating derivatives of the endoderm. Similar results wereobtained using several hESC lines (e.g., H1, H7, H9, and H13) culturedon the synthetic substrate for 1 month (6 passages).

Example 2 Separation of Pluripotent from Nonpluripotent Cells Culturedon Chemically Defined Surfaces Presenting GBPs

Methods:

GBP-presenting surfaces were produced essentially as described inExample 1. Pluripotent cells were cultured essentially as described inExample 1.

Effect of ROCK inhibitor on cellular adhesion: A mixture ofundifferentiated hESCs (Oct-4 positive) and differentiated cells derivedfrom embryoid bodies (Oct-4 negative) was seeded onto self-assembledmonolayer surfaces presenting the GBP GKKQRFRHRNRKG (SEQ ID NO: 1) andthe integrin binding peptide KGRGDS (SEQ ID NO: 5). For someexperiments, cells were seeded onto polyacrylamide gels or glasscoverslips presenting the CGKKQRFRHRNRKG (SEQ ID NO: 47) peptide. Thecells were grown for 24 hours in mTeSR medium containing 5 μM ROCKinhibitor Y-27632 and then switched to mTeSR medium without ROCKinhibitor for an additional 24 hours. The cells were fixed and stainedfor Oct-4 and counterstained with phalloidin and DAPI. In someinstances, cells were added to the surfaces without ROCK inhibitor.

Results:

As described in Example 1, pluripotent cells cultured on surfacespresenting GBPs in the presence of the ROCK inhibitor maintained highlevels of pluripotent cell-specific markers (FIG. 2B). However,significant detachment of undifferentiated cells was observed afterremoval of the ROCK inhibitor. In contrast, differentiated cellsremained attached and viable after the removal of ROCK inhibitor. Thus,removal of the ROCK inhibitor from the media selectively detachedundifferentiated, potentially teratoma-forming cells leaving apopulation of differentiated cells. When cells were added to thesurfaces without the ROCK inhibitor, some cells adhered to the surfacesbut few of these cells were Oct-4 positive undifferentiated cells.

In summary, the experiments described herein demonstrate that chemicallydefined, peptide-presenting surfaces having as little as a single typeof peptide can be used for routine culture and self-renewal ofpluripotent cells. In particular, SAMs presenting the peptides describedherein provided attachment, self-renewal, and colony spreading.

The invention has been described in connection with what are presentlyconsidered to be the most practical and preferred embodiments. However,the present invention has been presented by way of illustration and isnot intended to be limited to the disclosed embodiments. Accordingly,those skilled in the art will realize that the invention is intended toencompass all modifications and alternative arrangements within thespirit and scope of the invention as set forth in the appended claims.

The invention claimed is:
 1. A method for culturing pluripotent cells, the method comprising the step of: culturing pluripotent cells on an insoluble substrate having a chemically-defined surface that presents a GAG-binding peptide in a chemically defined culture medium comprising a Rho-associated kinase (ROCK) inhibitor, wherein the GAG-binding peptide is SEQ ID NO:1 or SEQ ID NO:2, and wherein the ROCK inhibitor is selected from the group consisting of (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]homopiperazine dihydrochloride (H-1152), 1-(5-isoquinolinesulfonyl)piperazine hydrochloride (HA-100), 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), 1-(5-isoquinolinesulfonyl)-3-methylpiperazine (iso H-7), N-2-(methylamino) ethyl-5-isoquinoline-sulfonamide dihydrochloride (H-8), N-(2-aminoethyl)-5-isoquinolinesulphonamide dihydrochloride (H-9), N-[2-p-bromo-cinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89), N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (HA-1004), 1-(5-isoquinolinesulfonyl)homopiperazine dihydrochloride (HA-1077), (S)-(+)-2-Methyl-4-glycyl-1-(4-methylisoquinolinyl-5-sulfonyl)homopiperazine dihydrochloride (glycyl H-1152) and (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride (Y-27632).
 2. The method as recited in claim 1, wherein the pluripotent cells are selected from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPS cells).
 3. The method as recited in claim 1, wherein the pluripotent cells are human cells.
 4. The method as recited in claim 1, wherein the peptide-presenting surface comprises an alkanethiol having about 3 to about 50 carbons.
 5. The method as recited in claim 4, wherein about 11 to about 18 carbons.
 6. The method as recited in claim 1, wherein the peptide occupies an area selected from the group consisting of between about 0.5% to about 100% of the surface, between about 0.5% to about 50% of the surface, and at least 30% of the surface.
 7. The method as recited in claim 1, wherein the ROCK inhibitor is (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride (Y27632). 